CN111713400A - Creation and identification method of cucumber-pickled cucumber two-body additional line - Google Patents
Creation and identification method of cucumber-pickled cucumber two-body additional line Download PDFInfo
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Abstract
The invention discloses a method for creating and identifying a cucumber-sour cucumber two-body additional line, belonging to the field of plant biotechnology breeding. The method comprises the steps of firstly backcrossing cucumber-sour cucumber interspecific hybridization allotetraploid and diploid cultivated cucumber twice to obtain a cucumber-sour cucumber monomer alien addition line, then selfing the cucumber-sour cucumber monomer alien addition line, and screening out a cucumber-sour cucumber two-body additional line by a molecular marking method and a genome in-situ hybridization method. The method has simple and convenient operation process and low cost, creates the cucumber-pickled cucumber dyadic body additional system material carrying the pickled cucumber No. 10 chromosome for the first time, and lays a foundation for excavating excellent genes of pickled cucumbers and further realizing genetic improvement of the cultivated cucumbers.
Description
Technical Field
The invention belongs to the field of plant biotechnology breeding, and particularly relates to a method for creating and identifying a cucumber-pickled cucumber two-body additional line, which can be used for transferring excellent genes of wild pickled cucumbers and genetic improvement of cultivated cucumber varieties.
Background
Cucumber (Cucumis sativus L.,2n ═ 2x ═ 14) originated in India and is a very important vegetable crop. So far, the high yield and the directional selection of cucumber have been pursued for a long time, which results in the single variety and narrower genetic base of cucumber (Staub et al, 1999), and the diseases and pests of cucumber pose serious threats to the production of cucumber, such as the spread of diseases like downy mildew and powdery mildew, and cause the large-area yield reduction of cucumber (bear et al, 2016; rainbow et al, 2017). Therefore, increasing the genetic diversity of the cucumber and breeding varieties with strong resistance to prevent disease threats are a safe and efficient way, and are also a necessary trend for the development of cucumber breeding.
Sour cucumber (Cucumis hystrix Chakr.,2n ═ 2x ═ 24) is a wild species of Cucumis melo in the origin Asia, carries multiple resistance genes including resistance to root-knot nematodes, powdery mildew, downy mildew, and the like, and has potential utility value (Perez-Garcia et al., 2009; Liu Xue et al, 2014). In cucumber, genetic resources are continuously reduced along with continuous utilization of germplasm resources, and variety improvement by wild resources is urgently needed, and excellent resistance genes of wild species can be introduced by interspecific hybridization to widen the genetic basis of cultivars, which is important for improvement of cucumber (Chen et al, 2003 a).
Interspecific crosses provide an important route for genetic improvement of crops by transferring resistance genes of wild species and increasing genetic variation (Kroon et al, 1979; Jena et al, 2016). However, because of the inter-species incompatibility, so far, in the melon genus, interspecific hybridization has been successfully achieved only between cucumber and pickled cucumber and, after further chromosome doubling, a new allotetraploid species Cucumis × hytivus Chen & Kirkride (HHCC,2n ═ 4x ═ 38) (Chen et al, 1997 b; Chen and Kirkride, 2000b), on the basis of which multiple allothromosomal materials are obtained by means of successive backcrosses (Chen et al, 2003 b; Chen et al, 2004; Cao Qing river, 2006; Zhangu et al, 2019).
Alien addition lines refer to lines formed by the addition of one or several foreign chromosomes to the genome of the recipient species, usually generated by backcrossing, and monomeric alien addition lines refer to lines formed by the addition of only one foreign chromosome in the background of the recipient genome (Gu et al 2015); a disomic line refers to an individual carrying a pair of homologous chromosomes from a foreign species. In addition to being An important material for studying the origin and evolution between species genomes and the chromosomal localization of superior genes (An et al, 2015; Li et al, 2016; Kong et al, 2018), disomic addition lines are genetically more stable than monomelic disomic addition lines, which are favorable for maintaining stability during meiosis, and are ideal bridge materials for genetic improvement of cucumbers and acceleration of breeding processes (wang feng et al, 2012). Therefore, the selection of the disomic addition line is important for transferring the wild excellent gene and accelerating the breeding process of the cucumber. However, the selection of the disomic addition line in Cucumis has not been reported so far.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for creating and identifying a cucumber-pickled cucumber two-body additional line, and the invention aims to provide an application of the method in cucumber breeding
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for preparing the disomic additional line of cucumber-sour cucumber includes such steps as backcrossing the allotetrad of interspecific hybridization between cucumber and sour cucumber with diploid cucumber to obtain the disomic additional line of cucumber-sour cucumber, selfing, molecular marking and in-situ hybridization of genome to obtain the disomic additional line of cucumber-sour cucumber.
A method for preparing a cucumber-pickled cucumber two-body additional line specifically comprises the following steps:
s1, backcrossing the cucumber with the allotetraploid material (C.hytivus) of the filial generation between the wild species of the same genus and the sour cucumber and the diploid cultivated cucumber (C.sativus) to create the allotriploid material;
s2, backcrossing the cucumber with diploid, and collecting seeds of mature fruits 40-60 days after pollination;
s3, screening a monomer heteroepisome line of the plant obtained after backcross by using a genome in-situ hybridization method, and identifying an exogenous chromosome in the monomer heteroepisome by using a molecular marker means;
s4, selfing the screened monomer alien addition lines, and continuously screening alien addition line plants through a molecular marking method for self-bred offspring;
s5, carrying out genome in situ hybridization on the selfing progeny of which the specific bands are amplified and determined by a molecular marking method to screen the disomic additional line, and taking the selfing progeny plant carrying two exogenous chromosomes of the pickled cucumber as the disomic additional line.
Further, the cucumber-sour cucumber interspecific hybridization allotetraploid genome is HHCC, H represents sour cucumber genome, C represents cucumber genome, and the number of chromosomes is 2 n-4 x-38.
In the method for identifying the cucumber-sour cucumber dyadic extra line, the cucumber-sour cucumber monomer extra line is subjected to selfing, and the selfed progeny firstly screens extra line plants by a molecular marker method; then screening out a heterozygosity plant carrying two exogenous chromosomes of the pickled cucumber by a genome in-situ hybridization method, namely a cucumber-pickled cucumber disomic additional line; the method specifically comprises the following steps:
1) extracting genome DNA of a cucumber-sour cucumber monomer heteroepisome self-bred progeny plant, carrying out PCR amplification on the genome DNA by using a primer pair for identifying a specific molecular marker of a sour cucumber exogenous chromosome, and judging the size of a target fragment according to the result of electrophoretic separation;
the exogenous chromosome of the pickled cucumber is a pickled cucumber 10 chromosome, a specific molecular marker SSH10 for identifying the pickled cucumber 10 chromosome is provided, and the nucleotide sequence of a specific molecular marker primer pair is as follows:
a forward primer: 5'-CCCTACAACTTCCCCTAT-3', respectively;
direction primers: 5'-TTCCTCTTCTTGGCTAAT-3' are provided.
2) Preparing mitosis chromosomes of plant materials which generate the cucumber chromosome specific bands by taking root tips of the mitosis vigorous parts, and selecting a slide with clear chromosomes and clean background to be detected;
dripping 100 mu L of 70% deionized formamide into the wafer to be detected, covering a glass slide, placing the wafer on a hybridization instrument at 80 ℃ for denaturation for 90s, immediately throwing off the glass slide after the denaturation is finished, and dehydrating the wafer for 5min by using 70%, 90% and 100% ethanol solutions in sequence at-20 ℃ and then airing; changing the hybridization mixture solution of 20 μ L in total in a metal bath at 90 deg.C for 6min, and then putting on ice for 10 min; dripping the denatured and hybridized mixed solution onto a glass slide which is denatured and dried, covering a cover glass, and placing the glass slide in a 37 ℃ hybridization instrument for hybridization overnight; after washing, the film is air-dried at room temperature, 20 mu L of 0.02mg/ml DAPI is added for counterstaining, and then the film is sealed by a cover glass, and the hybridization signal is observed by a fluorescence microscope under the condition of keeping out of the sun.
Further, in step 1), the PCR reaction system is 20 μ L, including 10 × PCR Buffer 2.0 μ L, 25mmol/LMgCl2mu.L of 1.2 mu.L, 2.0 mu.L of 150 mu mol/L dNTPs, 1.0 mu.L of each 0.67 mu mol/L labeled primer, 1 mu.L of 40 ng/mu L sample DNA, 0.2 mu.L of 5U/mu L Taq DNA polymerase and deionized water to make up to 20 mu.L;
the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, 29 cycles; extension at 72 ℃ for 10 min.
Further, in step 2), the hybridization mixture comprises 50% of deionized formamide, 2 XSSC, 10% of dextran sulfate and labeled probe.
Furthermore, the probe is a hy-gDNA probe, namely a pickled cucumber genome probe, and is used for identifying the number of exogenous chromosomes of pickled cucumber.
The preparation method is applied to cucumber breeding.
The identification method is applied to screening of cucumber-sour cucumber disomic addition lines for cucumber breeding.
Has the advantages that: compared with the prior art, the invention has the advantages that:
1) the invention creates a disomic addition line material in cucumis species for the first time, in particular to a cucumber-pickled cucumber disomic addition line carrying a pickled cucumber chromosome 10, and lays a foundation for digging excellent genes of pickled cucumbers and further realizing genetic improvement of cultivated cucumbers.
2) The invention screens the cucumber-sour cucumber disomic addition line by means of molecular markers and genome in-situ hybridization, and has simple and convenient operation process and low cost.
Drawings
Fig. 1 is a creation route diagram for the cucumber-gherkin dimer addition line;
FIG. 2 is a diagram of a cucumber-pickled cucumber monomer alien addition line carrying the No. 10 chromosome of pickled cucumber identified by using a pickled cucumber chromosome specific sequence marker SSH 10; in the figure, CC represents cultivated cucumber C.sativus, HH represents sour cucumber C.hystrix, HCC represents allotriploid, and CC-H10 represents cucumber-sour cucumber monomer addition line carrying sour cucumber chromosome 10;
FIG. 3 is the result of the amplification of the selfing progeny of the alien addition line of the monomer carrying the chromosome 10 of pickled cucumber with the specific sequence marker of the chromosome 10 of pickled cucumber: the inbred progeny with the specific strip shows to carry the chromosome 10 of the pickled cucumber; in the figure, CC denotes cultivated cucumber c.sativus; HH represents sour cucumber C.hystrix; HCC represents an allotriploid; 1-19 represent 19 surviving selfed progeny plants;
FIG. 4 is a diagram of a genomic in situ hybridization analysis of the cucumber-gherkin dimer addition line carrying chromosome 10 of Cucumis sativus (CC-2H 10); in the figure, hy-gDNA is used as a probe for identifying the number of exogenous chromosomes of the pickled cucumber;
FIG. 5 is a leaf-profile of the disomic addition line carrying chromosome 10 of Cucumis sativus.
Detailed Description
The invention is further described with reference to specific examples.
Example 1
The invention relates to a method for creating and identifying cucumber-pickled cucumber two-body additional line material carrying pickled cucumber chromosome 10, which comprises the following steps:
(1) creation of allotriploid: the cucumber obtained from old Jinfeng is backcrossed with diploid cucumber (C.sativus) to create an allotriploid material, wherein the allotriploid material is obtained by crossing between cucumber obtained from old Jinfeng and acid cucumber (Chen J F, Luo X D, Staub J E, et al, allotriploid derivative from a Amphiploid X diploid mating in Cucumis-I: Production, micropropagation and verification [ J ] Euphytoica, 2003,131(2):235 and 241).
(2) The creation of the cucumber-pickled cucumber monomer alien addition line: backcrossing the allotriploid and the diploid cultivar to obtain candidate alien addition line materials, screening the monomer alien addition lines by the candidate alien addition lines through genome in-situ hybridization, identifying the identity of an exogenous chromosome in the monomer alien addition lines by utilizing a pickled cucumber chromosome specific sequence marker, and finally screening the monomer alien addition line (CC-H10) carrying the pickled cucumber chromosome 10 as shown in figure 2.
(3) Creation of cucumber-pickled cucumber two-body additive line: selfing the selected disomic addition line carrying the chromosome 10 of the pickled cucumber to obtain 26 seeds, and finally surviving 19 plants of the materials after all the seeds are planted. To determine the chromosome composition of these 19 surviving selfed progeny plants, the selfed progeny plants were first PCR amplified with the pickled cucumber chromosome specific marker SSH 10. The cucumber chromosome specific marker primer is the following primer pair,
SSH 10: a forward primer: 5'-CCCTACAACTTCCCCTAT-3', respectively;
reverse primer: 5'-TTCCTCTTCTTGGCTAAT-3' are provided.
The PCR amplification method is as follows:
(1) the PCR reaction system is 20 mu L, and contains 2.0 mu L of 10 XPCR Buffer, 2.2 mu L of 25mmol/L MgCl21.2 mu L, 2.0 mu L of 150 mu mol/L dNTPs, 1.0 mu L of 0.67 mu mol/L labeled primers, 1 mu L of 40 ng/mu L genomic DNA, 0.2 mu L of 5U/mu L Taq DNA polymerase and deionized water to make up to 20 mu L;
(2) the amplification procedure was: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, 29 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;
(3) the PCR products were separated by electrophoresis on 2% agarose gel or 8% polyacrylamide gel.
As a result, as shown in fig. 3, 8 out of 19 selfed progeny plants produced cucumber chromosome-specific bands. To determine the number of chromosomes of gherkin in the 8 materials, genome in situ hybridization assays were performed on the 8 materials that produced the specific bands.
The genome in situ hybridization process is as follows:
(1) preparing mitosis chromosomes by taking root tips of the mitosis vigorous part, and selecting and storing the slide with clear chromosomes and clean background for later use under a phase contrast microscope after the preparation is finished;
(2) fluorescence in situ hybridization and signal detection: dripping 100 μ L of 70% deionized formamide into the wafer to be used, covering the glass slide, placing the wafer on a hybridization instrument at 80 ℃ for denaturation for 90s, immediately throwing off the glass slide after denaturation is finished, and dehydrating the wafer for 5min by using 70%, 90% and 100% ethanol solutions in sequence at-20 ℃ and then drying the wafer. A total of 20. mu.L of the hybridization mixture (containing 50% deionized formamide, 2 XSSC, 10% dextran sulfate and labeled probe) was denatured in a metal bath at 90 ℃ for 6min, and then placed on ice for 10min after denaturation. The denatured and mixed liquid is dropped on a glass slide which is denatured and dried in the air, and the glass slide is covered with a cover glass and placed in a 37 ℃ hybridization instrument for hybridization overnight. After washing, the slide was air-dried at room temperature, and 20. mu.L of DAPI (0.02mg/ml) was added to counterstain the slide, and after the slide was mounted, the hybridization signal was observed by an Olympus BX51 fluorescence microscope in the absence of light.
Finally, after genomic in situ hybridization analysis, as shown in fig. 4, two chromosomes showed fluorescent signals, carrying two gherkin chromosomes, a cucumber-gherkin dimer addition line (CC-2H 10).
Sequence listing
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Claims (9)
1. A method for creating the additional disomic line of cucumber-sour cucumber includes such steps as backcrossing the allotetrad of interspecific cross between cucumber and sour cucumber with diploid cucumber to obtain the allo-additional monoid line of cucumber-sour cucumber, selfing, molecular marking and in-situ hybridization of genome to obtain the additional disomic line of cucumber-sour cucumber.
2. A method of creating a cucumber-gherkin dimer line according to claim 1, comprising the steps of:
s1, backcrossing cucumber-sour cucumber interspecific hybridization allotetraploid and diploid cultivated cucumber to obtain allotriploid;
s2, backcrossing the allotriploid and the diploid cucumber to obtain a candidate alien addition line, and screening a monomer alien addition line through in-situ genome hybridization of the candidate alien addition line;
s3, selfing the screened monomer alien addition lines, and continuously screening alien addition line plants through a molecular marking method for self-bred offspring; then, the alien addition line plant carrying two exogenous chromosomes of the pickled cucumber is screened out by a genome in-situ hybridization method, namely the cucumber-pickled cucumber two-body additional line.
3. The method of claim 1 or 2, wherein the cucumber-sour cucumber disomic addition line has HHCC for cucumber-sour cucumber interspecific hybrid allotetraploid genome, H for sour cucumber genome, C for cucumber genome, and chromosome number 2 n-4 x-38.
4. The method of claim 1, wherein the cucumber-sour cucumber dyadic alien addition line is selfed, and the selfed progeny is first screened for alien addition line plants by molecular marker methods; then screening out a heterozygosity plant carrying two exogenous chromosomes of the pickled cucumber by a genome in-situ hybridization method, namely a cucumber-pickled cucumber disomic additional line; the method specifically comprises the following steps:
1) extracting genome DNA of a cucumber-sour cucumber monomer heteroepisome self-bred progeny plant, carrying out PCR amplification on the genome DNA by using a primer pair for identifying a specific molecular marker of a sour cucumber exogenous chromosome, and judging the size of a target fragment according to the result of electrophoretic separation;
the exogenous chromosome of the pickled cucumber is a pickled cucumber 10 chromosome, a specific molecular marker SSH10 for identifying the pickled cucumber 10 chromosome is provided, and the nucleotide sequence of a specific molecular marker primer pair is as follows:
a forward primer: 5'-CCCTACAACTTCCCCTAT-3', respectively;
reverse primer: 5'-TTCCTCTTCTTGGCTAAT-3' are provided.
2) Preparing mitosis chromosomes of plant materials which generate the cucumber chromosome specific bands by taking root tips of the mitosis vigorous parts, and selecting a slide with clear chromosomes and clean background to be detected;
dripping 100 mu L of 70% deionized formamide into the wafer to be detected, covering a glass slide, placing the wafer on a hybridization instrument at 80 ℃ for denaturation for 90s, immediately throwing off the glass slide after the denaturation is finished, and dehydrating the wafer for 5min by using 70%, 90% and 100% ethanol solutions in sequence at-20 ℃ and then airing; changing the hybridization mixture solution of 20 μ L in total in a metal bath at 90 deg.C for 6min, and then putting on ice for 10 min; dripping the denatured and hybridized mixed solution onto a glass slide which is denatured and dried, covering a cover glass, and placing the glass slide in a 37 ℃ hybridization instrument for hybridization overnight; after washing, the film is air-dried at room temperature, 20 mu L of 0.02mg/ml DAPI is added for counterstaining, and then the film is sealed by a cover glass, and the hybridization signal is observed by a fluorescence microscope under the condition of keeping out of the sun.
5. The method for identifying a cucumber-pickled cucumber dimeric episome as claimed in claim 4, wherein the PCR reaction system in step 1) is 20 μ L, comprising 10 × PCR Buffer 2.0 μ L and 25mmol/L MgCl2mu.L of 1.2 mu.L, 150 mu mol/L dNTPs 2.0 mu.L, 0.67 mu mol/L of labeled primer of 1.0 mu.L each, 40 ng/mu L of sample DNA of 1 mu.L, 5U/mu L of Taq DNA polymerase of 0.2 mu.L and deionized water to make up to 20 mu.L;
the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, 29 cycles; extension at 72 ℃ for 10 min.
6. The method of claim 4, wherein in step 2), the hybridization mixture comprises 50% deionized formamide, 2 XSSC, 10% dextran sulfate, and labeled probe.
7. The method of claim 6, wherein the probe is a hy-gDNA probe.
8. Use of the creation method of claim 1 in cucumber breeding.
9. Use of the identification method as claimed in claim 4 for screening cucumber-gherkin disomic addition lines for cucumber breeding.
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